Why worry about process-related impurities in C&GT development?

In response to the many cell and gene therapy (C&GTs) products in development [1], the FDA and EMA recently published new guidelines for developers advising greater monitoring of process-related impurities. However, many C&GT developers experience problems removing contaminants and properly characterizing the product.

What are impurities in complex biologics?

During the development and production of complex biologics such as cell & gene therapy products (C&GTs), process-related impurities co-purify along with the product. The EMA Q6B guidelines [2] divide these impurities into three subtypes depending on their origin:

• Cell substrate-derived (host cell proteins, host cell DNA, etc.)
  Cell culture-derived (antibiotics, IPTG, DTT, growth factors, etc.)
  Downstream-derived (enzymes, buffer components, etc.)
• The cell substrate-derived impurities include host cell proteins (HCPs), of which some are of concern if they are present in the final product. They include lipases, ubiquitin, peptidylprolyl isomerase, heat shock protein, cathepsin, and serine protease [3].

The cell culture-derived impurities include proteins added to the growth medium, e.g., fetal calf serum, human albumin, cytokines, and enzymes used for harvesting and lysing cells such as TrypLE.

Downstream-derived impurities consist of agents used to express and purify biological protein products. These include the Benzonase nuclease, affinity column antibodies for product capture, enzymes for site-specific PEGylation, aminopeptidase, and Protein A. Typical residuals also include Tris, carriers, ligands, Tween/Polysorbate, DCA, TCEP, heavy metals, solvents, Triton-X, antifoaming agents, PEI, TFA/Acetate, Imidazole, etc.

Why process-related impurities are a concern

Investigating residual protein levels is crucial, especially in the final product, because the residuals can influence the stability and efficacy of the active ingredient and may also pose a risk to the patient’s health:

Such ‘problematic’ proteins may…

1. Affect drug efficacy by reducing protein stability and potency,
   and/or
2. Induce immunotoxic effects and immunogenic reactions in patients
   -even in sub-ppm amounts.

The foreign, residual protein increases the risk of triggering an immune response in the patient.

Most host cell proteins (HCPs) have the potential to generate an immune response in humans due to the genomic differences between the human patient and commonly applied protein production hosts, such as E. coli, yeast, mouse myeloma cells (NS0), and Chinese Hamster Ovary cells (CHO). For example, Wang et al. showed that a reduction in HCPs correlated with a decline in the release of specific inflammatory cytokines [4].

There is also a risk that some unwanted HCPs, similar to human homologs, may have a biological function in humans resulting in potential side effects [4].

To summarize, it is essential to monitor residual proteins since they influence:

• The product quality by proteolysis, particle formation, or enzymatic modification;
• The process consistency and, therefore, the ability to produce a pure product;
• The patient safety by inducing immunogenicity or decreasing the activity of the biologic’s active ingredient.

With the development of sensitive analytical methods for residuals, the FDA and EMA are increasingly interested in data that describes the efficiency of the purification process for C&GTs.

Hence, an analytical method is needed to detect the exact process-related impurities in your product – throughout process development and the final drug substance.

It is challenging to monitor problematic residual proteins using ELISA

The FDA increasingly asks C&GT developers to document residual protein clearance before their biologic goes into late-stage clinical trials [5].

The problem is that traditional methods like ELISA and HPLC are limited by being unable to distinguish between impurities (low resolution) and insufficient sensitivity (limit of detection), respectively. Since the residuals are present at low-ppm levels, you need a reproducible approach with a high sensitivity to get a detailed overview of the residual protein clearance [6-7].

Furthermore, since the products are complex and contain proteins from multiple sources and species, a standard commercial ELISA cannot measure all these process-related impurities. An alternative is to develop a process-specific ELISA to cover the residual protein in these products, but this is costly, time-consuming, and challenging.

Orthogonal method for analysis of residual protein impurities

A typical challenge is the detection of protein residuals in products based on adenovirus expressed in a human cell line, e.g., HEK293 or A549 cells, grown on a cell substrate containing bovine serum albumin.